chapter 6 molecular biology of dna replication and recombination jones and bartlett publishers ©...
TRANSCRIPT
Chapter 6
Molecular Biology of DNA Replication and Recombination
Jones and Bartlett Publishers © 2005
A misinserted deoxynucleotide is excised by the proofreading exonuclease function
of DNA polymerase
Q1. Why is a mismatch a problem?
Q2. What is the consequence if proofreading doesn’t catch a mistake?
Addition of a dideoxynucleotide to the 3’-OH end of a DNA chain terminates chain elongation
The chain termination method of DNA sequencing
DNA sequencing machines use fluorescent dideoxynucleotides
A “shotgun sequencing” approach for sequencing of a large DNA molecule
Analogs of normal deoxynucleotides useful as anti-AIDS agents
Dideoxynucleosides are used because they can cross cell membranes more easily than can dideoxynucleotides
Excision repair of a mismatched base pair
Mismatch repair can result in ‘gene conversion’.
Variations from normal 2:2 segregation (gene conversion) can result from mismatch repair
Molecular details of recombination
• How are heteroduplexes formed as intermediate steps in recombination?
• First model that described molecular recombination was suggested by Holliday in 1964.
• Holliday junction
Symmetric model of homologous recombination showing some of the intermediate
steps and structures
Note the 2 nicks in the same location in the two duplexes in
step (B). This is followed by strand
exchange and Holliday junction
formation (C)
An asymmetric model of homologous recombination
Recombination is initiated by a cut in
only one of the 2 recombining duplexes
(A) followed by strand invasion (B)
and Holliday junction formation (C)
A recombining molecule showing the parental and the recombinant duplexes joined at the Holliday junction
Enzymes involved in recombination in E. coli
• Obvious ones: exonuclease, ligase
• Others: RecA
• RecBCD
• RuvC – Holliday junction-resolving enzyme
Resolution of a Holliday junction can occur by 2 alternative cleavage pathways
3D view of a Holliday junction
Double-strand break & repair
• The current favored model for homologous recombination is the double-strand break and repair model.
• A broken duplex interacts with an intact duplex.
• Gaps and sticky ends on broken duplex invade intact duplex.
• Displacement results (D loop).
Model for the recombinational repair of a double strand break in DNA